Cell culture device and method of using the same

ABSTRACT

A cell culture device includes: a holding member including a main body cylindrical portion extending in a cylindrical shape in an axial direction and a holding portion provided at an axially intermediate portion of the main body cylindrical portion and holding a culture membrane; a first cylindrical member detachably mounted on one axial side with respect to the holding member, extending in a cylindrical shape in the axial direction, and protruding from one axial end of the holding member toward the one axial side when being mounted to the holding member; and a second cylindrical member detachably mounted on another axial side with respect to the holding member, extending in a cylindrical shape in the axial direction, and protruding from another axial end of the holding member toward the other axial side when being mounted to the holding member.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based upon and claims the benefit of priority fromprior Japanese patent application No. 2019-065126, filed on Mar. 28,2019 and Japanese patent application No. 2019-065127, filed on Mar. 28,2019, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a cell culture device used forculturing cells and a method of using the same.

BACKGROUND ART

There is available a cell culture device for culturing cells on bothsurfaces of a culture membrane (for example, Patent Literature 1). Thecell culture device described in Patent Literature 1 includes a culturemembrane, an outer cylinder, and an inner cylinder. The outer cylinderincludes a main body cylindrical portion extending in a cylindricalshape in an axial direction. The main body cylindrical portion of theouter cylinder is provided with a slit extending in the axial direction.The outer cylinder is elastically deformable in a radial direction dueto the presence of the slit. The outer cylinder includes a holdingportion holding the culture membrane provided at an axial intermediateportion of the main body cylindrical portion.

Two inner cylinders are provided and each extend in a cylindrical shapein the axial direction. The first inner cylinder includes a facingportion facing the holding portion of the outer cylinder. The firstinner cylinder is inserted into the outer cylinder from one axial sideof the outer cylinder and is arranged adjacent to the outer cylinder. Inaddition, the second inner cylinder is inserted into the outer cylinderfrom the other axial side of the outer cylinder and is arranged adjacentto the outer cylinder. In a state in which each inner cylinder isinserted and mounted into the outer cylinder, the outer cylinder iselastically deformed radially outward, so that the inner cylinder andthe outer cylinder are in contact with each other in the radialdirection, and the facing portion of the first inner cylinder and theholding portion of the outer cylinder face each other in the axialdirection, so that the culture membrane is sandwiched and held.

Patent Literature 1: JP-A-2017-29092

In the cell culture device described above, the two inner cylinders areformed such that axial tip ends of the respective inner cylinders do notprotrude axially outward from axial end surfaces of the outer cylinderwhen mounted to the outer cylinder, respectively. Further, the outercylinder is formed in a relatively long axial length so that the twoinner cylinders can be mounted at the same time, and a dimensionaldistance from a lower end of the outer cylinder in the axial directionto the culture membrane in the cell culture device is long. Therefore,in order to culture cells in the culture membrane, it is necessary toincrease a volume of a well (culture vessel), and it is necessary toprepare a large amount of culture solution.

The present invention has been made in view of the above circumstances,and an object thereof is to provide a cell culture device capable ofculturing cells on a culture membrane with a small amount of culturesolution, and a method of using the same.

SUMMARY OF INVENTION

According to an aspect of the invention, there is provided a cellculture device comprising: a holding member including a main bodycylindrical portion extending in a cylindrical shape in an axialdirection and a holding portion provided at an axially intermediateportion of the main body cylindrical portion and holding a culturemembrane; a first cylindrical member detachably mounted on one axialside with respect to the holding member, extending in a cylindricalshape in the axial direction, and protruding from one axial end of theholding member toward the one axial side when being mounted to theholding member; and a second cylindrical member detachably mounted onanother axial side with respect to the holding member, extending in acylindrical shape in the axial direction, and protruding from anotheraxial end of the holding member toward the other axial side when beingmounted to the holding member.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an exploded perspective view of a cell culture deviceaccording to a first embodiment.

FIG. 2 is a sectional view of the cell culture device of the firstembodiment.

FIG. 3 is a sectional view for explaining a structure in which a culturemembrane is held by a holding member in the cell culture device of thefirst embodiment.

FIG. 4 is a sectional view of the cell culture device of the firstembodiment when cells are seeded on one axial end surface of the culturemembrane.

FIG. 5 is a sectional view of the cell culture device of the firstembodiment when cells are seeded on the other axial end surface of theculture membrane.

FIG. 6 is a flowchart showing an example of a procedure for using thecell culture device of the first embodiment during double-sided culture.

FIG. 7 is a sectional view of a cell culture device according to asecond embodiment.

FIG. 8 is an exploded perspective view of a holding member included inthe cell culture device of the second embodiment.

FIG. 9 is a sectional view of a cell culture device according to amodification.

FIG. 10 is an exploded perspective view of a cell culture deviceaccording to a third embodiment.

FIG. 11 is a side view (a partially sectional view) of the cell culturedevice of the third embodiment.

FIG. 12 is a top view of the cell culture device of the thirdembodiment.

FIG. 13 is a sectional view of the cell culture device of the thirdembodiment taken along a line IV-IV shown in FIG. 12.

FIG. 14 is a view for explaining locking portions of a first member anda second member included in the cell culture device of the thirdembodiment.

FIG. 15 is a view for explaining locking portions of a first member anda second member included in a cell culture device according to amodification.

FIG. 16 is a side view (left half) and a sectional view (right half) ofthe cell culture device according to a fourth embodiment.

FIG. 17 is a view showing a state before locking of locking portions ofa first member and a second member included in a cell culture deviceaccording to a fifth embodiment.

FIG. 18 is a view showing a state after the locking of the lockingportions of the first member and the second member included in the cellculture device according to the fifth embodiment.

DESCRIPTION OF EMBODIMENTS

Hereinafter, specific embodiments of a cell culture device and a methodof using the same according to the present invention will be describedwith reference to the drawings.

First Embodiment

A cell culture device 1 of a first embodiment is a device for culturinganchorage-dependent cells. The cell culture device 1 is capable ofculturing two types of cells on both surfaces of a membrane. As shown inFIG. 1, the cell culture device 1 includes a culture membrane 10, aholding member 20, a first cylindrical member 30, and a secondcylindrical member 40.

The culture membrane 10 is a sheet-like membrane on which cells arecultured. The culture membrane 10 includes a membrane body 11 and asupport frame 12. The membrane body 11 is a membrane main body portionserving as a scaffold for cells when cells are cultured. The membranebody 11 is a porous membrane provided with pores on both surfacesthereof. The membrane body 11 is formed of a thermosetting resin such aspolyurethane, silicone resin, phenol resin, epoxy resin, unsaturatedpolyester, or polyimide. A thickness of the membrane body 11 is, forexample, 0.1 μm to 100 μm.

The support frame 12 is a base material which supports and reinforcesthe membrane body 11. The support frame 12 is an annular frame bodyhaving a circular hole 12 a opened in a center portion thereof. Thesupport frame 12 is joined and fixed to a peripheral edge of themembrane body 11. Incidentally, a shape of the support frame 12 mayinclude at least an annular frame body portion, and may further includea protruding portion protruding radially outward from a part of theframe portion in a peripheral direction. The protruding portion has afunction for facilitating handling when a person grips the culturemembrane 10 with a jig such as a tweezer, for example. The support frame12 is formed of a material harder than the membrane body 11. Thematerial of the support frame 12 is, for example, a thermoplastic resinsuch as polyethylene, polypropylene, ABS resin, acrylic resin, andpolyethylene terephthalate (PET), or a thermosetting elastomer such assilicone rubber. A thickness of the support frame 12 is, for example,0.05 mm to 0.5 mm.

The holding member 20 is a member for holding the culture membrane 10.The holding member 20 is formed of a resin material, rubber, or thelike. The holding member 20 includes two holding cylinders, that is, afirst holding cylinder 21 and a second holding cylinder 22. The firstholding cylinder 21 and the second holding cylinder 22 are arrangedadjacent to each other in an axial direction X. The first holdingcylinder 21 and the second holding cylinder 72 hold the culture membrane10 in a state of being engaged with and fixed to each other. The culturemembrane 10 held by the holding member 20 is exposed on both axial sidesthrough axial openings of the first holding cylinder 21 and the secondholding cylinder 22.

The first holding cylinder 21 and the second holding cylinder 22 aremembers which are engageably attached to each other and have a structure23 by which the first holding cylinder 21 and the second holdingcylinder 22 are engaged with and fixed to each other as shown in FIG. 2.The engagement structure 23 is a locking structure which restrictsrelative movements between the first holding cylinder 21 and the secondholding cylinder 22 in the axial direction X and radial direction bywhich the first holding cylinder 21 and the second holding cylinder 22are separated from each other in an assembled state thereof. Theengagement structure 23 is provided at two or more positions in aperipheral direction (for example, three to five positions) in the firstholding cylinder 21 and the second holding cylinder 22. In addition, ina structure in which slits extending in the axial direction X arerespectively provided in the holding cylinders 21 22, the engagementstructure 23 is preferably provided at each of divided portionssandwiching the slit in the peripheral direction.

In a state in which the first holding cylinder 21 and the second holdingcylinder 22 are engaged with and fixed to each other as described above,a distance in which axial end surfaces of the first holding cylinder 21and the second holding cylinder 22 facing each other are separated inthe axial direction X is set to be equal to or less than an axialthickness of the culture membrane 10 (specifically, the support frame 12thereof) before assembling. Accordingly, the support frame 12 of theculture membrane 10 is sandwiched and fixed between the first holdingcylinder 21 and the second holding cylinder 22. At this time, gapsbetween the support frame 12 and the first holding cylinder 21 andbetween the support frame 12 and the second holding cylinder 22 aresealed.

The engagement structure 23 between the first holding cylinder 21 andthe second holding cylinder 22 may be, for example, an L-grooveengagement structure including a radially recessed concave portionprovided on an outer surface or an inner surface of one of the firstholding cylinder 21 and the second holding cylinder 22, and a radiallyprotruding convex portion provided on an inner surface or an outersurface of the other one. The concave portion may be, for example, agroove formed in an L shape when viewed from the radial direction.

The engagement structure 23 between the first holding cylinder 21 andthe second holding cylinder 22 may be, for example, a snap-fit typeengagement structure including an elastic deformation portionelastically deformable in the radial direction provided in one of thefirst holding cylinder 21 and the second holding cylinder 22, and aradially recessed groove portion provided on the other one. In thesnap-fit type engagement structure, when the first holding cylinder 21and the second holding cylinder 22 approach each other in the axialdirection when be assembled, the first holding cylinder 21 and thesecond holding cylinder 22 are engaged with and fixed to each other byfitting a radially protruding claw portion provided an axial tip endportion of the elastic deformation portion into the groove portion andbeing hooked.

The first holding cylinder 21 includes a hollow first cylindricalportion 21 a extending in a cylindrical shape in the axial direction X,a first holding portion 21 b which holds the culture membrane 10, and afirst side surface opening portion 21 c which is provided so as topenetrate the first cylindrical portion 21 a in the radial direction.The second holding cylinder 22 includes a hollow second cylindricalportion 22 a extending in a cylindrical shape in the axial direction X,a second holding portion 22 b which holds the culture membrane 10, and asecond side surface opening portion 22 c which is provided so as topenetrate the second cylindrical portion 22 a in the radial direction.

The first cylindrical portion 21 a and the second cylindrical portion 22a have respectively predetermined lengths La1, La2 in the axialdirection X. Incidentally, the axial lengths of the first cylindricalportion 21 a and the second cylindrical portion 22 a may substantiallycoincide with each other. The first cylindrical portion 21 a and thesecond cylindrical portion 22 a constitute a main body cylindricalportion extending in a cylindrical shape in the axial direction X of theholding member 20.

The first cylindrical portion 21 a and the second cylindrical portion 22a have respectively predetermined thicknesses in the radial direction.As shown in FIG. 3, outer diameters of the first cylindrical portion 21a and the second cylindrical portion 22 a substantially coincide witheach other, and inner diameters thereof substantially coincide with eachother. An outer diameter of the culture membrane 10 (that is, an outerdiameter of the support frame 12) exceeds the inner diameters of thefirst cylindrical portion 21 a and the second cylindrical portion 22 a.Incidentally, the outer diameter of the culture membrane 10 may besmaller than the outer diameters of the first cylindrical portion 21 aand the second cylindrical portion 22 a. The first holding cylinder 21and the second holding cylinder 22 sandwich and hold the culturemembrane 10 by engaging with each other in a state in which axial endsurfaces of the first cylindrical portion 21 a and the secondcylindrical portion 22 a facing in the axial direction X face eachother.

The first holding portion 21 b and the second holding portion 22 b arethe axial end surfaces of the first cylindrical portion 21 a and thesecond cylindrical portion 22 a which face each other, respectively. Theaxial end surfaces used as the first holding portion 21 b and the secondholding portion 22 b are abutment portions which can abut against thesupport frame 12 of the culture membrane 10. The first holding portion21 b abuts against an axial end surface of the culture membrane 10 onone axial side. The second holding portion 22 b abuts against the otheraxial end surface of the culture membrane 10 on the other axial side.Each of the first holding portion 21 b and the second holding portion 22b is formed in an annular shape. The first holding portion 21 b and thesecond holding portion 22 b are each provided at an axial intermediateportion of the main body cylindrical portion of the entire holdingmember 20.

The first side surface opening portion 21 c is a slit hole portion whichis provided so as to penetrate a predetermined portion of the firstcylindrical portion 21 a in the radial direction, and communicates theinside of the first cylindrical portion 21 a to the outside. The firstside surface opening portion 21 c is provided at one position in theperipheral direction. The predetermined portion in which the first sidesurface opening portion 21 c is provided includes at least a portion ona lower side in the axial direction X than the culture membrane 10 heldby the first holding portion 21 b during cell culture in a state inwhich the first cylindrical portion 21 a is positioned below the secondcylindrical portion 22 a, and is formed to extend from the first holdingportion 21 b to one axial side. The first side surface opening portion21 c extends over the entire axial direction of the first cylindricalportion 21 a. The first side surface opening portion 21 c has an airventing function of discharging air accumulated on a lower surface ofthe culture membrane 10 held by the first holding portion 21 b to theoutside.

The second side surface opening portion 22 c is a slit hole portionwhich is provided so as to penetrate a predetermined portion of thesecond cylindrical portion 22 a in the radial direction, andcommunicates the inside of the second cylindrical portion 22 a to theoutside. The second side surface opening portion 22 c is provided at oneposition in the peripheral direction. The predetermined portion in whichthe second side surface opening portion 22 c is provided includes atleast a portion on the lower side in the axial direction X than theculture membrane 10 held by the second holding portion 22 b during thecell culture in a state in which the second cylindrical portion 22 a ispositioned below the first cylindrical portion 21 a, and is formed toextend from the second holding portion 22 b to the other axial side. Thesecond side surface opening portion 22 c extends over the entire axialdirection of the second cylindrical portion 22 a. The second sidesurface opening portion 22 c has an air venting function of dischargingair accumulated on the lower surface of the culture membrane 10 held bythe second holding portion 22 b to the outside.

Each of the first cylindrical member 30 and the second cylindricalmember 40 is a cylindrical member attached to the holding member 20. Thefirst cylindrical member 30 and the second cylindrical member 40 areeach formed of a resin material, rubber, or the like. The firstcylindrical member 30 and the second cylindrical member 40 areselectively attached to the holding member 20 when cells are cultured onthe culture membrane 10. That is, the first cylindrical member 30 isattached to the holding member 20 when first cells are seeded on oneaxial end surface of the culture membrane 10, while the secondcylindrical member 40 is attached to the holding member 20 when secondcells are seeded on the other axial end surface of the culture membrane10.

The first cylindrical member 30 includes a hollow cylindrical portion 31extending in a cylindrical shape in the axial direction X. The firstcylindrical member 30 is mounted on one axial side of the holding member20 (specifically, the first holding cylinder 21). The first cylindricalmember 30 can be attached to/detached from the holding member 20(specifically, the first holding cylinder 21). That is, the firstcylindrical member 30 and the first holding cylinder 21 are engageablyattached to each other. The first cylindrical member 30 and the firstholding cylinder 21 have a structure A by which the first cylindricalmember 30 and the first holding cylinder 21 are engaged with and fixedto each other, such as the engagement structure 23 of the first holdingcylinder 21 and the second holding cylinder 22 of the holding member 20.The culture membrane 10 held by the holding member 20 is exposed to oneaxial side through an axial opening of the first cylindrical member 30.

The first cylindrical member 30 is formed so as to be insertable intothe first holding cylinder 21 of the holding member 20. The firstcylindrical member 30 is attached in a state of being inserted into thefirst cylindrical portion 21 a of the first holding cylinder 21. Thefirst cylindrical member 30 includes an outer diameter which coincideswith the inner diameter of the first holding cylinder 21. Thecylindrical portion 31 of the first cylindrical member 30 includes afirst protruding portion 32 which protrudes from one axial end of thefirst holding cylinder 21 toward the one axial side in a state of beingengaged with and fixed to the first holding cylinder 21.

Specifically, the first cylindrical member 30 is formed such that anaxial end surface of the cylindrical portion 31 on an insertion sideabuts against the one axial end surface of the culture membrane 10sandwiched and fixed between the first holding cylinder 21 and thesecond holding cylinder 22, and an axial end surface of the cylindricalportion 31 on an opposite side to the insertion side is arranged at aposition protruding toward one axial side from the one axial end of thefirst holding cylinder 21, in the state in which the first cylindricalmember 30 is engaged with and fixed to the first holding cylinder 21.That is, a length Lb1 of the first cylindrical member 30 in the axialdirection X is set to be larger than a length La1 of the firstcylindrical portion 21 a of the first holding cylinder 21 in the axialdirection X.

The second cylindrical member 40 includes a hollow cylindrical portion41 extending in a cylindrical shape in the axial direction X. The secondcylindrical member 40 is mounted on the other axial side of the holdingmember 20 (specifically, the second holding cylinder 22). The secondcylindrical member 40 can be attached to/detached from the holdingmember 20 (specifically, the second holding cylinder 22). That is, thesecond cylindrical member 40 and the second holding cylinder 22 areengageably attached to each other. The second cylindrical member 40 andthe second holding cylinder 22 have a structure B by which the secondcylindrical member 40 and the second holding cylinder 22 are engagedwith and fixed to each other, such as the engagement structure 23 of thefirst holding cylinder 21 and the second holding cylinder 22 of theholding member 20. The culture membrane 10 held by the holding member 20is exposed to the other axial side through an axial opening of thesecond cylindrical member 40.

The second cylindrical member 40 is formed so as to be insertable intothe second holding cylinder 22 of the holding member 20. The secondcylindrical member 40 is attached in a state of being inserted into thesecond cylindrical portion 22 a of the second holding cylinder 22. Thesecond cylindrical member 40 includes an outer diameter which coincideswith the inner diameter of the second holding cylinder 22. The secondcylindrical portion 41 of the second cylindrical member 40 includes asecond protruding portion 42 which protrudes from the other axial end ofthe second holding cylinder 22 to the other axial side in a state ofbeing engaged with and fixed to the second holding cylinder 22.

Specifically, the second cylindrical member 40 is formed such that anaxial end surface of the cylindrical portion 41 on the insertion sideabuts against the other axial end surface of the culture membrane 10sandwiched and fixed between the first holding cylinder 21 and thesecond holding cylinder 22, and an axial end surface of the cylindricalportion 41 on an opposite side to the insertion side is arranged at aposition protruding toward the other axial side from the other axial endof the second holding cylinder 22, in the state in which the secondcylindrical member 40 is engaged with and fixed to the second holdingcylinder 22. That is, a length Lb2 of the second cylindrical member 40in the axial direction X is set to be larger than a length La2 of thesecond cylindrical portion 22 a of the second holding cylinder 22 in theaxial direction X.

Next, a method of assembling the cell culture device 1 and a method ofusing the same will be described with reference to FIGS. 4, 5, and 6.

The culture membrane 10, the holding member 20, the first cylindricalmember 30, and the second cylindrical member 40, which are components ofthe cell culture device 1, are prepared. Incidentally, the culturemembrane 10 and the holding member 20 may be those in which the culturemembrane 10 is held by the first holding cylinder 21 and the secondholding cylinder 22 of the holding member 20 being engaged with andfixed to each other.

The cell culture device 1 is assembled manually by a person using a jigsuch as a tweezer. In an assembling procedure, first, the firstcylindrical member 30 is assembled to the holding member 20 holding theculture membrane 10. First, the first cylindrical member 30 is grippedby a jig in a state in which the holding member 20 is set such that anaxial end portion side thereof from which the first cylindrical member30 is inserted is positioned upward, then the first cylindrical member30 is inserted into the holding member 20 by being moved from above in adirection approaching each other in the axial direction X, andthereafter, the holding member 20 and the first cylindrical member 30are engaged with and fixed to each other by the structure A, therebyrealizing the assembling.

When such assembling is performed, the first cylindrical member 30 ismounted to the holding member 20, and the first protruding portion 32protrudes outward from the one axial end of the first holding cylinder21 of the holding member 20 toward the one axial side (a state shown inFIG. 4 step S100 shown in FIG. 6). The cell culture on the culturemembrane 10 is performed on the cell culture device 1 in this state. Inthe cell culture, the cell culture device 1 may be placed in a well of aplate for the cell culture, filled with a culture solution, and seededwith cells α on the one axial end surface (upper surface shown in FIG.4) of the culture membrane 10 (specifically, the membrane body 11). Theseeding of the cells α is performed by first immersing the cell culturedevice 1 in the culture solution with the one axial end surface of theculture membrane 10 facing vertically upward, and in this state,supplying the cells α to a space on the one axial end surface side (stepS110).

After the seeding of the cells α is completed as described above, thefirst cylindrical member 30 is removed from the holding member 20 usinga jig (step S120), and then the holding member 20 is turned upside downwhile holding the culture membrane 10 (step S130).

After the holding member 20 is turned upside down, the secondcylindrical member 40 is assembled to the holding member 20. First, thesecond cylindrical member 40 is gripped by a jig in a state in which theholding member 20 is set such that an axial end portion side thereoffrom which the second cylindrical member 40 is inserted, which is anopposite side to the axial end portion side into which the firstcylindrical member 30 is inserted, is positioned upward, then the secondcylindrical member 40 is inserted into the holding member 20 by beingmoved from above in a direction approaching each other in the axialdirection X, and thereafter, the holding member 20 and the secondcylindrical member 40 are engaged with and fixed to each other by thestructure B, thereby realizing the assembling.

When such assembling is performed, the second cylindrical member 40 ismounted to the holding member 20, and the second protruding portion 42protrudes outward from the other axial end of the second holdingcylinder 22 of the holding member 20 toward the other axial side (astate shown in FIG. 5; step S140). The cell culture on the culturemembrane 10 is performed on the cell culture device 1 in this state. Inthe cell culture, the cell culture device 1 may be placed in a well of aplate for the cell culture, filled with a culture solution, and seededwith cells 13 on the other axial end surface (upper surface shown inFIG. 5) of the culture membrane 10 (specifically, the membrane body 11).The seeding of the cells α is performed by first immersing the cellculture device 1 in the culture solution with the other axial endsurface of the culture membrane 10 facing vertically upward, and in thisstate, supplying the cells β to a space on the other axial end surfaceside (step S150).

After the seeding of the cells 13 is completed as described above, thesecond cylindrical member 40 is removed from the holding member 20 byusing a jig (step S160). Then, the culture membrane 10 on which the cellculture is performed is removed from the holding member 20, andmicroscopic observation or the like of the membrane body 11 of theculture membrane 10 is performed.

As described above, according to the structure of the cell culturedevice 1, two types of cells can be seeded on both surfaces of theculture membrane 10 to perform the cell culture. In addition, when twotypes of cells are seeded on both surfaces of the culture membrane 10,in both cases of seeding cells on the one axial end surface of theculture membrane 10 and seeding cells on the other axial end surfacethereof, it is necessary to mount one of the first cylindrical member 30and the second cylindrical member 40 to an upper side of the holdingmember 20 holding the culture membrane 10, but it is not necessary tomount the other one of the first cylindrical member 30 and the secondcylindrical member 40 to a lower side of the holding member 20. That is,when two types of cells are seeded on both surfaces of the culturemembrane 10, the cell culture device 1 can be used in a state ofremoving either of the first cylindrical member 30 and the secondcylindrical member 40 which is positioned on the lower side of theholding member 20 from the holding member 20 without requiring both thefirst cylindrical member 30 and the second cylindrical member 40 to bemounted to the holding member 20 and them to be used at the same time.

Since the length Lb1 of the first cylindrical member 30 in the axialdirection X is larger than the length La1 of the first cylindricalportion 21 a of the first holding cylinder 21 in the axial direction X,the first cylindrical member 30 protrudes from the one axial end of thefirst holding cylinder 21 toward the one axial side at the firstprotruding portion 32 in the state of being engaged with and fixed tothe first holding cylinder 21. Since the length Lb2 of the secondcylindrical member 40 in the axial direction X is larger than the lengthLa2 of the second cylindrical portion 22 a of the second holdingcylinder 22 in the axial direction X, the second cylindrical member 40protrudes from the other axial end of the second holding cylinder 22toward the other axial side at the second protruding portion 42 in thestate of being engaged and fixed to the second holding cylinder 22.Therefore, according to the cell culture device 1, since a dimensionaldistance from an axial lower end to the holding portions 21 b, 22 bholding the culture membrane 10 can be shortened when the cell cultureis performed, a volume of the well can be reduced, and cells can becultured on the culture membrane with a small amount of culturesolution.

In the cell culture device 1, the first holding cylinder 21 of theholding member 20 includes the first side surface opening portion 21 cpenetrating the first cylindrical portion 21 a in the radial direction.The second holding cylinder 22 of the holding member 20 includes thesecond side surface opening portion 22 c penetrating the secondcylindrical portion 22 a in the radial direction. The side surfaceopening portions 21 c, 22 c are provided at the predetermined portionsincluding the portions on the lower side in the axial direction X thanthe culture membrane 10 held by the holding portions 21 b, 22 b,respectively. Therefore, air accumulated on both surfaces of the culturemembrane 10 can be discharged to the outside of the holding member 20via the side surface opening portions 21 c, 22 c.

In particular, after cells are seeded on the one axial end surface ofthe culture membrane 10 held by the holding member 20, when the holdingmember 20 is turned upside down and cells are seeded on the other axialend surface of the culture membrane 10, air accumulated on the one axialend surface of the culture membrane 10 on which cell seeding has alreadybeen performed can be discharged to the outside of the holding member 20via the side surface opening portion 21 c. Therefore, it is possible toprevent air from remaining on a surface on which the cells are initiallyseeded, and it is possible to avoid inconvenience caused by the airremaining (for example, cells which are initially seeded do not comeinto contact with the culture solution, or the like).

In the cell culture device 1, the culture membrane 10 includes themembrane body 11 on which cells are seeded, and the support frame 12joined to the membrane body 11 and formed of a material harder than themembrane body 11. The culture membrane 10 is sandwiched and fixedbetween the first holding cylinder 21 and the second holding cylinder 22of the holding member 20 by the support frame 12. Therefore, since thesupport frame 12 which is relatively hard may be used for handling theculture membrane 10 with a jig, the handling of the culture membrane 10can be facilitated. In addition, the support frame 12 is formed in anannular shape by being joined to the peripheral edge of the membranebody 11. Therefore, evaluation such as the microscopic observation ofthe membrane body 11 of the culture membrane 10 can be performed whilethe support frame 12 is joined to the membrane body 11.

Further, since the first holding cylinder 21 and the second holdingcylinder 22 are engaged with and fixed to each other, the holding member20 sandwiches and holds the culture membrane 10 by the first holdingcylinder 21 and the second holding cylinder 22. Therefore, the holdingmember 20 can be easily assembled while holding the culture membrane 10,and the holding member 20 after assembling can be easily separated intothe first holding cylinder 21 and the second holding cylinder 22, sothat the culture membrane 10 can be easily taken out from the holdingmember 20. As a result, the culture membrane 10 after the cell culturecan be independently evaluated by the microscopic observation or thelike, and the evaluation can be easily and appropriately performed ascompared with a structure in which the culture membrane and the holdingmember are integrated.

In the first embodiment, the first holding portion 21 b of the firstholding cylinder 21 and the second holding portion 22 b of the secondholding cylinder 22 each correspond to a “holding portion” described inthe claims, respectively.

In the first embodiment, two types of cells are seeded on both surfacesof the culture membrane 10. However, the present invention is notlimited to this, and three or more types of cells may be seeded on bothsurfaces of the culture membrane 10. For example, three types of cellsmay be prepared, two types of cells may be seeded on one surface of theculture membrane 10, and then the culture membrane 10 may be turnedupside down, and the other one type of cells may be seeded on the othersurface of the culture membrane 10.

Second Embodiment

Similarly to the cell culture device 1 of the first embodiment, a cellculture device 100 of a second embodiment can culture two types of cellson both surfaces of the membrane. As shown in FIG. 7, the cell culturedevice 100 includes the culture membrane 10, a holding member 120, afirst cylindrical member 130, and a second cylindrical member 140.Incidentally, in the second embodiment, the same components as those ofthe cell culture device 1 in the first embodiment are denoted by thesame reference numerals, and a description thereof will be omitted orsimplified.

The holding member 120 is a member for holding the culture membrane 10.The holding member 120 is formed of a resin material, rubber, or thelike. As shown in FIG. 8, the holding member 120 includes two holdingcylinders, and has a first holding cylinder 121 and a second holdingcylinder 122. The first holding cylinder 121 and the second holdingcylinder 122 are arranged adjacent to each other in the axial directionX. The first holding cylinder 121 and the second holding cylinder 122hold the culture membrane 10 in a state of being engaged with and fixedto each other. The culture membrane 10 held by the holding member 120 isexposed on both axial sides through axial openings of the first holdingcylinder 121 and the second holding cylinder 122.

The first holding cylinder 121 and the second holding cylinder 122 aremembers which are engageably attached to each other, and have astructure in which the first holding cylinder 121 and the second holdingcylinder 122 are engaged with and fixed to each other. The engagementstructure may be an L-groove engagement structure or a snap-fit typeengagement structure similar to the engagement structure 23 of theholding member 20 in the first embodiment. In a state in which the firstholding cylinder 121 and the second holding cylinder 122 are engagedwith and fixed to each other, a distance in which holding portions ofthe first holding cylinder 121 and the second holding cylinder 122facing each other are separated in the axial direction X is set to besmaller than the axial thickness of the culture membrane 10(specifically, the support frame 12 thereof) before assembling.Accordingly, the support frame 12 of the culture membrane 10 issandwiched and fixed between the first holding cylinder 121 and thesecond holding cylinder 122. At this time, gaps between the supportframe 12 and the first holding cylinder 121 and between the supportframe 12 and the second holding cylinder 122 are sealed.

The first holding cylinder 121 includes a hollow first cylindricalportion 121 a extending in a cylindrical shape in the axial direction X,a first holding portion 121 b which holds the culture membrane 10, and afirst side surface opening portion 121 c provided so as to penetrate thefirst cylindrical portion 121 a in the radial direction. The secondholding cylinder 122 includes a hollow second cylindrical portion 122 aextending in a cylindrical shape in the axial direction X, a secondholding portion 122 b which holds the culture membrane 10, a second sidesurface opening portion 122 c and a third side surface opening portion122 d provided so as to penetrate the second cylindrical portion 122 ain the radial direction, and a holding opening portion 122 e provided soas to penetrate the second holding portion 122 b in the axial directionX.

The first cylindrical portion 121 a has a predetermined length in theaxial direction X and has a predetermined thickness in the radialdirection. The second cylindrical portion 122 a has a predeterminedlength in the axial direction X and has a predetermined thickness in theradial direction. An outer diameter of the first cylindrical portion 121a and an inner diameter of the second cylindrical portion 122 asubstantially coincide with each other. The outer diameter of theculture membrane 10 (that is, the outer diameter of the support frame12) exceeds an inner diameter of the first cylindrical portion 121 a andis equal to or less than the outer diameter of the first cylindricalportion 121 a, and is equal to or less than the inner diameter of thesecond cylindrical portion 122 a.

The first holding portion 121 b is an axial end surface facing thesecond holding cylinder 122 in the first cylindrical portion 121 a. Thesecond holding portion 122 b is a flange portion protruding radiallyinward from the second cylindrical portion 122 a. The outer diameter ofthe culture membrane 10 (that is, the outer diameter of the supportframe 12) is larger than an inner diameter of a radially inner end ofthe second holding portion 122 b. The second holding portion 122 b isprovided at an axial intermediate portion of the second cylindricalportion 122 a. The first holding cylinder 121 is engaged with and fixedto the second holding cylinder 122 in a state in which the firstcylindrical portion 121 a is inserted inside the second cylindricalportion 122 a of the second holding cylinder 122 and the first holdingportion 121 b faces the second holding portion 122 b. Accordingly, thefirst holding cylinder 121 and the second holding cylinder 122 sandwichand hold the culture membrane 10.

The first side surface opening portion 121 c is a slit hole portionwhich is provided so as to penetrate a predetermined portion of thefirst cylindrical portion 121 a in the radial direction, andcommunicates the inside of the first cylindrical portion 121 a to theoutside. The first side surface opening portion 121 c is provided at oneposition in the peripheral direction. The predetermined portion in whichthe first side surface opening portion 121 c is provided includes atleast a portion on the lower side in the axial direction X than theculture membrane 10 held by the first holding portion 121 b during thecell culture in a state in which the first cylindrical portion 121 a ispositioned below the second cylindrical portion 122 a, and is formed toextend from the first holding portion 121 b to one axial side. The firstside surface opening portion 121 c has an air venting function ofdischarging air accumulated on the lower surface of the culture membrane10 held by the first holding portion 121 b to the outside in a state inwhich the first cylindrical portion 121 a is positioned below the secondcylindrical portion 122 a.

Each of the second side surface opening portion 122 c and the third sidesurface opening portion 122 d is a slit hole portion which is providedso as to penetrate a predetermined portion of the second cylindricalportion 122 a in the radial direction, and communicates the inside ofthe second cylindrical portion 122 a to the outside. The second sidesurface opening portion 122 c and the third side surface opening portion122 d are provided at different positions of the second cylindricalportion 122 a in the peripheral direction. As a result, a torsionalrigidity of the second holding cylinder 122 can be improved and asealing property can be improved.

The predetermined portion in which the second side surface openingportion 122 c is provided includes at least a portion on the lower sidein the axial direction X than the culture membrane 10 held by the secondholding portion 122 b during the cell culture in a state in which thesecond cylindrical portion 122 a is positioned below the firstcylindrical portion 121 a, and is formed to extend from the secondholding portion 122 b to the other axial side. The second side surfaceopening portion 122 c has an air venting function of discharging airaccumulated on the lower surface of the culture membrane 10 held by thesecond holding portion 122 b to the outside in a state where the secondcylindrical portion 122 a is positioned below the first cylindricalportion 121 a.

The predetermined portion in which the third side surface openingportion 122 d is provided includes at least a portion which radiallycommunicates with the first side surface opening portion 121 c of thefirst holding cylinder 121 and is on the lower side in the axialdirection X than the culture membrane 10 held by the second holdingportion 122 b during the cell culture in the state in which the firstcylindrical portion 121 a is positioned below the second cylindricalportion 122 a, and is formed to extend from the second holding portion122 b to the one axial side. The third side surface opening portion 122d has an air venting function which communicates with the first sidesurface opening portion 121 c and discharges air accumulated on thelower surface of the culture membrane 10 held by the second holdingportion 122 b to the outside in the state in which the first cylindricalportion 121 a is positioned below the second cylindrical portion 122 a.

The holding opening portion 122 e is provided so as to penetrate thesecond holding portion 122 b in the axial direction X, and communicateswith the second side surface opening portion 122 c in the radialdirection. The holding opening portion 122 e has an air venting functionwhich communicates with the second side surface opening portion 122 cand discharges air accumulated on the lower surface of the culturemembrane 10 held by the second holding portion 122 b to the outside inthe state in which the second cylindrical portion 122 a is positionedbelow the first cylindrical portion 121 a.

Each of the first cylindrical member 130 and the second cylindricalmember 140 is a cylindrical member attached to the holding member 120.The first cylindrical member 130 and the second cylindrical member 140are each formed of a resin material, rubber, or the like. The firstcylindrical member 130 and the second cylindrical member 140 areselectively attached to the holding member 20 when cells are cultured onthe culture membrane 10. That is, the first cylindrical member 130 isattached to the holding member 120 when the first cells are seeded onone axial end surface of the culture membrane 10, while the secondcylindrical member 140 is attached to the holding member 120 when thesecond cells are seeded on the other axial end surface of the culturemembrane 10.

The first cylindrical member 130 includes a hollow cylindrical portion131 extending in a cylindrical shape in the axial direction X. The firstcylindrical member 130 is mounted on one axial side of the holdingmember 120 (specifically, the first holding cylinder 121). The firstcylindrical member 130 can be attached to/detached from the firstholding cylinder 121. The first cylindrical member 130 and the firstholding cylinder 121 have a structure in which the first cylindricalmember 130 and the first holding cylinder 121 are engaged with and fixedto each other. The culture membrane 10 held by the holding member 120 isexposed to one axial side through an axial opening of the firstcylindrical member 130.

The first cylindrical member 130 is formed so as to be insertable intothe first holding cylinder 121 of the holding member 120. The firstcylindrical member 130 is attached in a state of being inserted into thefirst cylindrical portion 121 a of the first holding cylinder 121. Thefirst cylindrical member 130 is formed such that an inner diameterthereof is invariable over the entire area in the axial direction X, butan outer diameter thereof on the other axial side is smaller than anouter diameter thereof on one axial side. The outer diameter on theother axial side coincides with the inner diameter of the first holdingcylinder 121. The cylindrical portion 131 of the first cylindricalmember 130 includes a first protruding portion 132 which protrudes fromone axial end of the first holding cylinder 121 toward the one axialside in a state of being engaged with and fixed to the other axial sideof the first holding cylinder 121.

The first cylindrical member 130 is formed such that an axial endsurface of the cylindrical portion 131 on the insertion side abutsagainst the one axial end surface of the culture membrane 10 sandwichedand fixed between the first holding cylinder 121 and the second holdingcylinder 122, and an axial end surface of the cylindrical portion 131 onan opposite side to the insertion side is arranged at a positionprotruding toward the one axial side from the one axial end of the firstholding cylinder 121, in the state in which the first cylindrical member130 is engaged with and fixed to the first holding cylinder 121. Thatis, a length of the first cylindrical member 130 in the axial directionX is set to be larger than a length of the first cylindrical portion 121a of the first holding cylinder 121 in the axial direction X.

The second cylindrical member 140 includes a hollow cylindrical portion141 extending in a cylindrical shape in the axial direction X. Thesecond cylindrical member 140 is mounted on the other axial side of theholding member 120 (specifically, the second holding cylinder 122). Thesecond cylindrical member 140 can be attached to/detached from thesecond holding cylinder 122. The second cylindrical member 140 and thesecond holding cylinder 122 have a structure in which the secondcylindrical member 140 and the second holding cylinder 122 are engagedwith and fixed to each other. The culture membrane 10 held by theholding member 120 is exposed to the other axial side through an axialopening of the second cylindrical member 140.

The second cylindrical member 140 is formed to be insertable into thesecond holding cylinder 122 of the holding member 120. The secondcylindrical member 140 is attached in a state of being inserted into thesecond cylindrical portion 122 a of the second holding cylinder 122. Thesecond cylindrical member 140 has an outer diameter which coincides withthe inner diameter of the second cylindrical portion 122 a of the secondholding cylinder 122. The second cylindrical portion 141 of the secondcylindrical member 140 includes a second protruding portion 142 Whichprotrudes from the other axial end of the second holding cylinder 122 tothe other axial side in a state of being engaged with and fixed to thesecond holding cylinder 122.

The second cylindrical member 140 is formed such that an axial endsurface of the cylindrical portion 141 on the insertion side abutsagainst the other axial end surface of the culture membrane 10sandwiched and fixed between the first holding cylinder 121 and thesecond holding cylinder 122 and abuts against the second holding portion122 b having a flange shape, and an axial end surface of the cylindricalportion 141 on an opposite side to the insertion side is arranged at aposition protruding toward the other axial side from the other axial endof the second holding cylinder 122, in the state in which the secondcylindrical member 140 is engaged with and fixed to the second holdingcylinder 122. That is, a length of the second cylindrical member 140 inthe axial direction X is set to be larger than a length of the secondcylindrical portion 122 a of the second holding cylinder 122 in theaxial direction X.

As an assembling procedure of the cell culture device 100 having theabove structure, first, the first cylindrical member 130 is assembled tothe holding member 120 holding the culture membrane 10, as in the caseof the cell culture device 1 of the first embodiment. Then, the cellsare seeded on the one axial end surface of the culture membrane 10 in astate in which the first protruding portion 132 protrudes outward fromthe one axial end of the first holding cylinder 121 of the holdingmember 120 toward the one axial side. After the cell seeding iscompleted, the first cylindrical member 130 is removed from the holdingmember 120, and then the holding member 120 is turned upside down whileholding the culture membrane 10.

Next, the second cylindrical member 140 is assembled to the holdingmember 120. Then, the cells are seeded on the other axial end surface ofthe culture membrane 10 in a state in which the second protrudingportion 142 protrudes outward from the other axial end of the secondholding cylinder 122 of the holding member 120 toward the other axialside. After the cell seeding is completed, the second cylindrical member140 is removed from the holding member 120, and then the microscopicobservation or the like of the membrane body 11 of the culture membrane10 is performed.

As described above, even in the structure of the cell culture device100, two types of cells can be seeded on both surfaces of the culturemembrane 10 to perform the cell culture. In addition, when two types ofcells are seeded on both surfaces of the culture membrane 10, the cellculture device 100 can be used in a state of removing either of thefirst cylindrical member 130 and the second cylindrical member 140 whichis positioned on the lower side of the holding member 120 withoutrequiring both the first cylindrical member 130 and the secondcylindrical member 140 to be mounted and used at the same time. Thefirst cylindrical member 130 and the second cylindrical member 140protrude outward in the axial direction X from the axial ends of theholding member 120 by the protruding portions 132, 142 in a state ofbeing engaged with and fixed to the holding member 120, respectively.Therefore, according to the cell culture device 100, since a dimensionaldistance from an axial lower end to the holding portions 121 b, 122 bholding the culture membrane 10 can be shortened when the cell cultureis performed, the volume of the well can be reduced, and cells can becultured on the culture membrane with a small amount of culturesolution.

In the cell culture device 100, the first holding cylinder 121 of theholding member 120 includes the first side surface opening portion 121 cpenetrating the first cylindrical portion 121 a in the radial direction.The second holding cylinder 122 of the holding member 120 includes thesecond side surface opening portion 122 c and the third side surfaceopening portion 122 d penetrating the second cylindrical portion 122 ain the radial direction and the holding opening portion 122 epenetrating the second holding portion 122 b in the axial direction X.The side surface opening portions 121 c, 122 c and 122 d are provided atthe respective predetermined portions including portions on the lowerside in the axial direction X than the culture membrane 10 held by theholding portions 121 b, 122 b when cells are cultured on both surfacesof the culture membrane 10, respectively. In particular, the third sidesurface opening portion 122 d is provided so as to expose the lowersurface of the culture membrane 10 held by the holding portions 121 b,122 b to the outside of the second holding cylinder 122 via the firstside surface opening portion 121 c when cells are seeded on the otheraxial end surface of the culture membrane 10. In addition, the holdingopening portion 122 e is provided so as to expose the lower surface ofthe culture membrane 10 held by the holding portions 121 b, 122 b to theoutside of the second holding cylinder 122 via the second side surfaceopening portion 122 c when cells are seeded on the one axial end surfaceof the culture membrane 10. Therefore, air accumulated on both surfacesof the culture membrane 10 can be discharged to the outside of theholding member 120 via the side surface opening portions 121 c, 122 c.

In the second embodiment, the first holding portion 121 b of the firstholding cylinder 121 and the second holding portion 122 b of the secondholding cylinder 122 each correspond to the “holding portion” describedin the claims, respectively.

In the first and second embodiments described above, the cell culturedevices 1, 100 are used in which the cylindrical members 30, 40, 130,and 140 are inserted and arranged inside cylinders of the holdingmembers 20, 120. However, the present invention is not limited to this,and as shown in FIG. 9, the present invention may be applied to a cellculture device 200 in which cylindrical members 230, 240 are insertedand arranged outside a cylinder of a holding member 220.

In the first and second embodiments described above, both of the twoholding cylinders 21, 22 constituting the holding member 20 and both ofthe holding cylinders 121, 122 constituting the holding member 120 arerespectively provided with the opening portions 21 c, 22 c, 121 c, 122c, 122 d, and 122 e which discharge air accumulated on the lower surfaceof the culture membrane 10 to the outside. However, the presentinvention is not limited to this, and only in order to discharge airaccumulated on the one axial end surface of the culture membrane 10 tothe outside of the holding members 20, 120 when cells are seeded on theother axial end surface of the culture membrane 10 after the holdingmembers 20, 120 are turned upside down, a holding cylinder 221 of twoholding cylinders 221, 222 positioned below when cells are seeded on theother axial end surface of the culture membrane 10 may be provided withan opening portion 221 c for discharging air accumulated on the lowersurface of the culture membrane 10 to the outside as shown in FIG. 9.

In the first and second embodiments described above, the culturemembrane 10 includes the membrane body 11 and the annular support frame12 joined to the peripheral edge of the membrane body 11. However, thepresent invention is not limited to this, and the support frame 12 maybe formed in, for example, a cross shape or a net shape when viewed fromthe axial direction X as long as the support frame 12 has a frame-likestructure joined to the membrane body 11.

In the first and second embodiments described above, the culturemembrane 10 includes the membrane body 11 and the support frame 12joined to the peripheral edge of the membrane body 11. However, thepresent invention is not limited to this, and as shown in FIG. 9, theculture membrane 210 may be formed only of a membrane body having nosupport frame. In this modification, the culture membrane 210 may bejoined and fixed to either of the first holding cylinder 221 and thesecond holding cylinder 222 of the holding member 220. In thismodification, an outer diameter of the culture membrane 210 may belarger than inner diameters of the first holding cylinder 221 and thesecond holding cylinder 222 and equal to or less than outer diameters ofthe first holding cylinder 221 and the second holding cylinder 222. Inaddition, in this modification, it is preferable that peripheralsurfaces between the holding cylinders 221, 222 and cylindrical members230, 240 are sealed in order to ensure an inside/outside sealingproperty of an periphery of the first holding cylinder 221 holding theculture membrane 210 and an inside/outside sealing property of anperiphery of the second holding cylinder 222 holding the culturemembrane 210 when cells are cultured.

Further, in the first and second embodiments described above, thecylindrical members 30, 40, 130, and 140 are formed such that the axialend surfaces thereof on the insertion side abut against the supportframe 12 of the culture membrane 10 when the cylindrical members 30, 40,130, and 140 are mounted to the holding members 20, 120, respectively.However, the present invention is not limited to this, and the axial endsurfaces of the cylindrical members 30, 40, 130, and 140 may not abutagainst the support frame 12 of the culture membrane 10 when thecylindrical members 30, 40, 130, and 140 are mounted to the holdingmembers 20, 120, respectively.

Third Embodiment

-   A cell culture device 1001 of a third embodiment is a device for    culturing anchorage-dependent cells. The cell culture device 1001 is    capable of culturing two types of cells on both surfaces of a    membrane. As shown in FIGS. 10, 11, 12, and 13, the cell culture    device 1001 includes a culture membrane 1010, a first member 1020,    and a second member 1030. In the cell culture device 1001, the    culture membrane 1010 is sandwiched and fixed between the first    member 1020 and the second member 1030.

The culture membrane 1010 is a sheet-like membrane on which cells arecultured. The culture membrane 1010 includes a membrane body 1011 and asupport frame 1012. The membrane body 1011 is a membrane main bodyportion serving as a scaffold for cells when cells are cultured. Themembrane body 1011 is a porous membrane provided with pores on bothsurfaces thereof. The membrane body 1011 is formed of a thermosettingresin such as polyurethane, silicone resin, phenol resin, epoxy resin,unsaturated polyester, or polyimide. A thickness of the membrane body1011 is, for example, 0.1 μm to 110 μm.

The support frame 1012 is a base material which supports and reinforcesthe membrane body 1011. The support frame 1012 is an annular frame bodyhaving a circular hole 1012 a opened in a center portion thereof. Thesupport frame 1012 is joined and fixed to a peripheral edge of themembrane body 1011. Incidentally, a shape of the support frame 1012 mayinclude at least an annular frame body portion, and may further includea protruding portion protruding radially outward from a part of theframe portion in a peripheral direction. The protruding portion has afunction for facilitating handling when a person grips the culturemembrane 1010 with a jig such as a tweezer, for example. The supportframe 1012 is formed of a material harder than the membrane body 1011.The material of the support frame 1012 is, for example, a thermoplasticresin such as polyethylene, polypropylene, ABS resin, acrylic resin, andpolyethylene terephthalate (PET), or a thermosetting elastomer such assilicone rubber. A thickness of the support frame 1012 is, for example,0.05 mm to 0.5 mm.

The first member 1020 is a base material for sandwiching and fixing theculture membrane 1010. The first member 1020 is a member formed in acylindrical shape. The first member 1020 is formed of a resin material,rubber, or the like. The first member 1020 includes a first cylindricalportion 1021, a placing portion 1022, and a side surface opening portion1023. The first cylindrical portion 1021 and the placing portion 1022are formed integrally with each other.

The first cylindrical portion 1021 is a hollow portion extending in acylindrical shape in the axial direction X. The first cylindricalportion 1021 is formed in a cylindrical shape, for example.Incidentally, an inner diameter of the first cylindrical portion 1021may be constant regardless of a position thereof in the axial directionX, or may vary (more specifically, be larger as the position in theaxial direction X is farther from the placing portion 1022) depending onthe position thereof in the axial direction X. The first cylindricalportion 1021 includes a peripheral wall portion 1021 a. An innerperipheral surface of the peripheral wall portion 1021 a is formed to beable to guide the culture membrane 1010 in the axial direction X to theplacing portion 1022.

The peripheral wall portion 1021 a includes a cutout portion 1021 b. Thecutout portion 1021 b penetrates the peripheral wall portion 1021 a inthe radial direction and extends in the axial direction X. The cutoutportion 1021 b extends in the axial direction X from an axial endportion of the first cylindrical portion 1021 where the culture membrane1010 is inserted into the first member 1020 in the axial direction X toa portion where the placing portion 1022 is provided. The cutout portion1021 b is provided in a portion of the peripheral wall portion 1021 a inthe peripheral direction, and is provided at one position on theperipheral wall portion 1021 a. The cutout portion 1021 b is a slit forinserting a jig for attaching and detaching the culture membrane 1010sandwiched between the first member 1020 and the second member 1030 toand from the placing portion 1022 from the radial outside. That is, thecutout portion 1021 b has a function of allowing the jig inserted fromthe radial outside to the first member 1020 to be movable in the axialdirection X.

The placing portion 1022 is a portion on which the culture membrane 1010is placed. The placing portion 1022 is connected to one axial end of thefirst cylindrical portion 1021. The placing portion 1022 is formed in anannular shape on an inner peripheral side of one axial end of the firstcylindrical portion 1021. Incidentally, the placing portion 1022 may beprovided intermittently in the peripheral direction on the innerperipheral side of the one axial end of the first cylindrical portion1021. The placing portion 1022 includes a wall portion 1022 a on whichan axial end surface facing in the axial direction X is formed, and acircular hole portion 1022 b which is opened at a central portion of thewall portion 1022 a. The placing portion 1022 abuts against the supportframe 1012 of the culture membrane 1010 at an inner axial end surface ofthe wall portion 1022 a to hold the culture membrane 1010. The culturemembrane 1010 held on the wall portion 1022 a of the placing portion1022 is exposed to the outside through the hole portion 1022 b of theplacing portion 1022.

The side surface opening portion 1023 is provided so as to penetrate theperipheral wall portion 1021 a of the first cylindrical portion 1021 inthe radial direction. The side surface opening portion 1023 is a holeportion which allows the inside of the first cylindrical portion 1021 tocommunicate with the outside. The side surface opening portion 1023 isprovided at a portion of the peripheral wall portion 1021 a in theperipheral direction, and is provided at a predetermined portion of theperipheral wall portion 1021 a. The predetermined portion at which theside surface opening portion 1023 is provided includes at least aportion closer to the placing portion 1022 side in the axial direction Xthan the culture membrane 1010 placed on the placing portion 1022. Theside surface opening portion 1023 has an air venting function ofdischarging air accumulated on the lower surface of the culture membrane1010 placed on the placing portion 1022 to the outside. The side surfaceopening portion 1023 is provided at the same peripheral position as thecutout portion 1021 b in the first member 1020. The side surface openingportion 1023 is arranged so as to be continuous with the cutout portion1021 b in the axial direction X, and is integrated with the cutoutportion 1021 b.

The second member 1030 is paired with the first member 1020 and a basematerial for sandwiching and fixing the culture membrane 1010. Thesecond member 1030 is a member formed in a cylindrical shape. The secondmember 1030 is inserted and arranged in the first cylindrical portion1021 of the first member 1020. The second member 1030 is formed of aresin material, rubber, or the like. The second member 1030 includes asecond cylindrical portion 1031, a facing portion 1032, and a grippingportion 1033. The second cylindrical portion 1031, the facing portion1032, and the gripping portion 1033 are formed integrally with eachother.

The second cylindrical portion 1031 is a hollow portion extending in acylindrical shape in the axial direction X. The second cylindricalportion 1031 is formed in a cylindrical shape, for example. An outerdiameter of the second cylindrical portion 1031 has a size whichsubstantially coincides with the inner diameter of the first cylindricalportion 1021 of the first member 1020. Incidentally, an inner diameteror the outer diameter of the second cylindrical portion 1031 may beconstant regardless of a position thereof in the axial direction X, ormay vary depending on the position thereof in the axial direction X(more specifically, be larger as the position in the axial direction Xis farther from the placing portion 1022).

The facing portion 1032 is a portion facing the wall portion 1022 a ofthe placing portion 1022 of the first member 1020 in the axial directionX. The facing portion 1032 has a function of sandwiching the culturemembrane 1010 with the placing portion 1022 of the first member 1020.The facing portion 1032 is connected to one axial end of the secondcylindrical portion 1031. The facing portion 1032 is formed in anannular shape on an inner peripheral side of the one axial end of thesecond cylindrical portion 1031. Incidentally, the facing portion 1032may be provided intermittently in the peripheral direction on the innerperipheral side of the one axial end of the second cylindrical portion1031.

The facing portion 1032 includes a wall portion 1032 a on which an axialend surface facing in the axial direction X is formed, and a circularhole portion 1032 b which is opened at a central portion of the wallportion 1032 a. The facing portion 1032 abuts against the support frame1012 of the culture membrane 1010 at an outer axial end surface of thewall portion 1032 a to hold the culture membrane 1010. The culturemembrane 1010 sandwiched between the placing portion 1022 and the facingportion 1032 is exposed to an inner space of the second cylindricalportion 1031 through the hole portion 1032 b of the facing portion 1032.

The gripping portion 1033 has a function of facilitating handling of thesecond member 1030 when a person assembles the second member 1030 to thefirst member 1020 with a jig such as a tweezer. The gripping portion1033 includes an arm portion 1033 a and a connection portion 1033 b. Aplurality of (for example, three) arm portions 1033 a are provided. Oneend of each of the arm portions 1033 a is connected to the other axialend of the second cylindrical portion 1031 on an opposite side to thefacing portion 1032 in the axial direction. The arm portions 1033 a arearranged at intervals in the peripheral direction of the secondcylindrical portion 1031. The arm portion 1033 a extends outward in theaxial direction X from the other axial end of the second cylindricalportion 1031. The connection portion 1033 b is formed in an annularshape so as to connect the other ends of the plurality of arm portions1033 a. The connection portion 1033 b is cut out at a part in theperipheral direction, and is formed in a C shape.

The first member 1020 and the second member 1030 each have a lockingportion which restricts a relative movement between the first member1020 and the second member 1030 in the axial direction X by which thefirst member 1020 and the second member 1030 are separated from eachother in an assembled state. That is, the first member 1020 includes aconcave portion 1025 as a locking portion, and the second member 1030includes a convex portion 1035 as the locking portion. As shown in FIG.14, the concave portion 1025 and the convex portion 1035 restrict therelative movement between the first member 1020 and the second member1030 in the axial direction X by which the first member 1020 and thesecond member 1030 are separated from each other in the assembled stateof the first member 1020 to the second member 1030.

The concave portion 1025 of the first member 1020 is a groove portionprovided on an inner surface of the peripheral wall portion 1021 a ofthe first cylindrical portion 1021 so as to be recessed radiallyoutward. The concave portions 1025 are provided at two peripheralpositions (for example, different positions by 180°) of the first member1020, for example. The concave portion 1025 is formed in an L shape.That is, the concave portion 1025 includes an axial parallel portion1025 a extending in the axial direction X and an orthogonal portion 1025b extending in a direction orthogonal to the axial direction X. Theaxial parallel portion 1025 a and the orthogonal portion 1025 b eachhave a width greater than or equal to a width of the convex portion 1035of the second member 1030. The axial parallel portion 1025 a extendsfrom the other axial end on an opposite side to the placing portion 1022of the first cylindrical portion 1021 to the one axial end side. Theorthogonal portion 1025 b is connected to one axial end portion of theaxial parallel portion 1025 a and extends in the orthogonal direction.The orthogonal portion 1025 b forms a locking wall 1026 on the otheraxial end side to Which the convex portion 1035 of the second member1030 is locked.

The convex portion 1035 of the second member 1030 is a protrudingportion provided on an outer surface of the second cylindrical portion1031 so as to protrude radially outward. The convex portions 1035 areprovided at two peripheral positions (for example, different positionsby) 180° of the second member 1030, for example. The convex portion 1035is formed to have a size corresponding to the concave portion 1025(particularly, the orthogonal portion 1025 b). Specifically, the convexportion 1035 has a width (that is, an axial length) corresponding to thewidth (that is, an axial length) of the orthogonal portion 1025 b of theconcave portion 1025.

Incidentally, the width of the orthogonal portion 1025 b of the concaveportion 1025 may vary depending on a peripheral position thereof. Forexample, as shown in FIG. 15, the width of the orthogonal portion 1025 bmay be the widest on a side close to the axial parallel portion 1025 asuch that the convex portion 1035 is easily moved from the side close tothe axial parallel portion 1025 a to an inner side far from the axialparallel portion 1025 a in the orthogonal portion 1025 b when the firstmember 1020 and the second member 1030 are assembled, and may begradually narrowed toward the inner side far from the axial parallelportion 1025 a. In addition, the width of the orthogonal portion 1025 bis set to a width of a predetermined position on the inner side of theorthogonal portion 1025 b at which the convex portion 1035 is fittedbeing wider than the minimum width position, so that the predeterminedposition is closer to the axial parallel portion 1025 a than the minimumwidth position. Therefore, the convex portion 1035 is difficultly movedfrom the predetermined position to the side close to the axial parallelportion 1025 a after the convex portion 1035 is fitted at thepredetermined position.

The assembling of the first member 1020 and the second member 1030 isperformed such that the convex portion 1035 moves along the concaveportion 1025. The convex portion 1035 can be fitted into the vicinity ofa tip end side of the concave portion 1025 and locked to the lockingwall 1026. The first member 1020 and the second member 1030 areassembled to each other by fitting the convex portion 1035 into theconcave portion 1025 and being locked to the locking wall 1026, therebyrestricting the relative movement between the first member 1020 and thesecond member 1030 in the axial direction X by which the first member1020 and the second member 1030 are separated from each other.

In a state in which the assembling of the first member 1020 and thesecond member 1030 is completed by fitting the convex portion 1035 intothe concave portion 1025, a distance between the placing portion 1022and the facing portion 1032 in the axial direction X is set to besmaller than the thickness of the support frame 1012 of the culturemembrane 1010 before the assembling. Therefore, in a state in which theassembling is completed, the culture membrane 1010 is sandwiched betweenthe placing portion 1022 of the first member 1020 and the facing portion1032 of the second member 1030.

Next, assembling of the cell culture device 1001 will be described.

The culture membrane 1010, the first member 1020, and the second member1030, which are components of the cell culture device 1001, areprepared. The cell culture device 1001 is assembled manually by a personusing a jig such as a tweezer. In an assembling procedure, first, theculture membrane 1010 is assembled to the first member 1020. The culturemembrane 1010 is gripped with a jig, the culture membrane 1010 is theninserted into the first member 1020 by being moved in the axialdirection X from the opposite side to the placing portion 1022 of thefirst member 1020 in the axial direction, and thereafter, the culturemembrane 1010 is placed so as to abut against the axial end surface ofthe placing portion 1022 of the first member 1020, thereby realizing theassembling of the culture membrane 1010 to the first member 1020.Incidentally, the assembling of the culture membrane 1010 to the firstmember 1020 described above may be performed in a state in which thefirst member 1020 is set with the axial end portion thereof on a placingportion 1022 side being positioned below.

Next, as described above, the second member 1030 is assembled to thefirst member 1020 on which the culture membrane 1010 is placed on theplacing portion 1022. First, the gripping portion 1033 of the secondmember 1030 is gripped by a jig, the second member 1030 is then insertedinto the first member 1020 by being moved in a direction approachingeach other in the axial direction X from the opposite side to theplacing portion 1022 of the first member 1020 in the axial direction,the convex portion 1035 of the second member 1030 is moved along theaxial parallel portion 1025 a of the recess 1025 of the first member1020, and thereafter, the second member 1030 is moved in one peripheraldirection with respect to the first member 1020, and the convex portion1035 of the second member 1030 is moved along the orthogonal portion1025 b of the concave portion 1025 of the first member 1020, therebyrealizing the assembling. Incidentally, the assembling of the secondmember 1030 to the first member 1020 described above may be performed byinserting the second member 1030 from above the first member 1020 in astate in which the first member 1020 is set with the axial end portionthereof on the placing portion 1022 side being positioned below.

When the movement is performed and the convex portion 1035 of the secondmember 1030 is fitted into the vicinity of the tip end side of theconcave portion 1025 of the first member 1020, the relative movement ofthe two members 1020, 1030 in the axial direction X by which the twomembers 1020, 1030 are separated from each other is restricted, and thetwo members 1020, 1030 are mounted to each other, so that the assemblingof the two members 1020, 1030 is completed. When the assembling of thetwo members 1020, 1030 is completed, the placing portion 1022 and thefacing portion 1032 sandwich the culture membrane 1010 (specifically,the support frame 1012) therebetween, and the convex portion 1035 of thesecond member 1030 is locked to the locking wall 1026 of the concaveportion 1025 of the first member 1020.

According to the structure of the cell culture device 1001, after thefirst member 1020 and the second member 1030 are assembled, the relativemovement between the first member 1020 and the second member 1030 in theaxial direction X can be restricted by the concave portion 1025 and theconvex portion 1035 as the locking portions in a state in which thesupport frame 1012 of the culture membrane 1010 is sandwiched betweenthe first member 1020 and the second member 1030. Therefore,adhesiveness in the assembled state of the first member 1020 and thesecond member 1030 sandwiching the culture membrane 1010 can beimproved, and a sealing property on both sides sandwiching the culturemembrane 1010 during cell culture can be ensured. Although the culturemembrane 1010 positioned in the side surface opening portion 1023 is notsandwiched between the first member 1020 and the second member 1030,since an area is narrow, the culture membrane 1010 is sealed only by anelastic pressing force of the support frame 1012 and the second member1030.

After the assembling of the first member 1020 and the second member 1030is completed as described above, the cell culture is performed on onesurface of the culture membrane 1010. In the cell culture, the cellculture device 1001 may be placed in a well of a plate for cell cultureand be filled with a culture solution. The cell culture on the onesurface of the culture membrane 1010 is performed by first immersing thecell culture device 1001 in the culture solution with one surface of theculture membrane 1010 on the second member 1030 side facing verticallyupward, and in this state, supplying first cells to a space on onesurface side.

When the culture of the first cells is completed as described above, theassembling of the first member 1020 and the second member 1030 of thecell culture device 1001 is released, and the first member 1020 and thesecond member 1030 are assembled again by turning the culture membrane1010 upside down. The releasing of the assembling of the first member1020 and the second member 1030 is performed in a reverse procedure tothe assembling procedure described above. Specifically, the releasing ofthe assembling is realized by first moving the second member 1030 in theother peripheral direction with respect to the first member 1020, andthen moving the second member 1030 in a direction away from the firstmember 1020 in the axial direction X. When the assembling is released,next, the culture membrane 1010 is moved in the axial direction X by thejig from the state in which the culture membrane 1010 is placed on theplacing portion 1022 of the first member 1020, is then turned upsidedown, and is placed again so as to abut against the axial end surface ofthe placing portion 1022. Further, as described above, the second member1030 is assembled to the first member 1020 on which the culture membrane1010 is placed on the placing portion 1022.

When the assembling is completed, the placing portion 1022 and thefacing portion 1032 sandwich the culture membrane 1010 therebetween, andthe convex portion 1035 of the second member 1030 is locked to thelocking wall 1026 of the concave portion 1025 of the first member 1020,so that the same effect as described above can be obtained. After theassembling is completed, the cell culture is performed on the othersurface of the culture membrane 1010. The cell culture on the othersurface of the culture membrane 1010 is performed by first immersing thecell culture device 1001 in the culture solution with the other surfaceof the culture membrane 1010 on the second member 1030 side facingvertically upward, and in this state, supplying second cells to a spaceon the other surface side.

As described above, according to the structure of the cell culturedevice 1001, two types of cells can be cultured on both surfaces of theculture membrane 1010. In addition, when two types of cells are culturedon both surfaces of the culture membrane 1010, after the cells arecultured on one side of the culture membrane 1010, the culture membrane1010 itself may be turned upside down after the assembling of the firstmember 1020 and the second member 1030 is released, and it is notnecessary to turn the first member 1020 and the second member 1030upside down. Therefore, it is possible to improve a workability inculturing two types of cells on both surfaces of the culture membrane1010.

In addition, when two types of cells are cultured on both surfaces ofthe culture membrane 1010, it is not necessary to make the cell culturedevice vertically symmetrical in consideration of the upside down of theculture membrane 1010. Therefore, a structure of the first member 1020on the placing portion 1022 side in the axial direction X with respectto the culture membrane 1010 placed on the placing portion 1022 can besimplified, the cell culture device 1001 can be reduced in a size, and asize of the well and the amount of the culture solution can be reducedin realizing the cell culture. Further, since the culture membrane 1010can be removed from the first member 1020 and the second member 1030independently, the culture membrane 1010 after the cell culture can beindependently evaluated by the microscopic observation or the like, andthe evaluation can be easily and appropriately performed as comparedwith a structure in which the culture membrane and a holding member areintegrated.

In the cell culture device 1, the first member 1020 includes the cutoutportion 1021 b which penetrates the peripheral wall portion 1021 a ofthe first cylindrical portion 1021 in the radial direction and extendsin the axial direction X. The cutout portion 1021 b is a slit forinserting a jig such as a tweezer from the radial outside of the firstmember 1020 and moving the jig in the axial direction X. The culturemembrane 1010 is placed on the placing portion 1022 of the first member1020 and is sandwiched between the first member 1020 and the secondmember 1030, and can be gripped by a jig such as a tweezer inserted fromthe cutout portion 1021 b for attaching and detaching the culturemembrane 1010 to and from the placing portion 1022. Therefore, theculture membrane 1010 can be easily attached to and detached from theplacing portion 1022 of the first member 1020 using a jig, and the cellculture can be performed on both surfaces of the culture membrane 1010with a minimum configuration without requiring many components as thecell culture device 1001 to be prepared for realizing the cell cultureon both sides of the culture membrane 1010.

The first member 1020 includes the side surface opening portion 1023which penetrates the peripheral wall portion 1021 a of the firstcylindrical portion 1021 in the radial direction. The side surfaceopening portion 1023 is provided at the predetermined portion of theperipheral wall portion 1021 a including at least a portion closer tothe placing portion 1022 side in the axial direction X than the culturemembrane 1010 placed on the placing portion 1022. Therefore, after thefirst cells are cultured on the one surface of the culture membrane1010, when the culture membrane 1010 is turned upside down and thesecond cells are cultured on the other surface thereof, air accumulatedon a surface on which the first cells are cultured can be discharged tothe outside of the first member 1020 via the side surface openingportion 1023, so that it is possible to prevent air from remaining onthe surface on which the first cells are initially seeded, and it ispossible to avoid inconvenience caused by the air remaining (forexample, the first cells do not come into contact with the culturesolution, or the like).

The cutout portion 1021 b and the side surface opening portion 1023described above are provided at the same position in the peripheraldirection on the first member 1020, and are arranged so as to becontinuous with each other in the axial direction X and integrated witheach other. Therefore, a mold structure of the first member 1020provided with the cutout portion 1021 b and the side surface openingportion 1023 can be simplified, and the first member 1020 can bemanufactured accurately and easily.

In the third embodiment, the concave portion 1025 of the first member1020 and the convex portion 1035 of the second member 1030 eachcorrespond to a “locking portion” described in the claims.

In the third embodiment, the cutout portion 1021 b and the side surfaceopening portion 1023 of the first member 1020 are provided at the sameposition in the peripheral direction, and are arranged so as to becontinuous with each other in the axial direction X. However, thepresent invention is not limited to this, and the cutout portion 1021 band the side surface opening portion 1023 of the first member 1020 maybe provided at positions different from each other in the peripheraldirection, and may be arranged so as not to be continuous with eachother in the axial direction X. According to a structure of thismodification, a torsional rigidity of the first member 1020 in theperipheral direction can be ensured as compared with the structure ofthe third embodiment.

In the third embodiment, the first member 1020 includes the concaveportion 1025 provided on the inner surface of the peripheral wallportion 1021 a of the first cylindrical portion 1021 so as to berecessed radially outward, and the second member 1030 includes theconvex portion 1035 provided on the outer surface of the secondcylindrical portion 1031 so as to protrude radially outward. However,the present invention is not limited to this, and the first member 1020may include a convex portion provided on the inner surface of the firstcylindrical portion 1021 so as to protrude radially inward, and thesecond member 1030 may include a concave portion provided on the outersurface of the second cylindrical portion 1031 so as to be recessedradially outward.

In the third embodiment, two types of cells are seeded on both surfacesof the culture membrane 1010. However, the present invention is notlimited to this, and three or more types of cells may be seeded on bothsurfaces of the culture membrane 1010. For example, three types of cellsmay be prepared, two types of cells may be seeded on one surface of theculture membrane 1010, and then the culture membrane 1010 may be turnedupside down, and the other one type of cells may be seeded on the othersurface of the culture membrane 1010.

Fourth Embodiment

Similarly to the cell culture device 1001 of the third embodiment, acell culture device 1100 of a fourth embodiment is a device forculturing anchorage-dependent cells. Hereinafter, in the cell culturedevice 1100, the same components as those of the cell culture device1001 are denoted by the same reference numerals, and a descriptionthereof will be omitted or simplified.

As shown in FIG. 16, the cell culture device 1100 includes the culturemembrane 1010, the first member 1110, and the second member 1120. In thecell culture device 1100, the culture membrane 1010 is sandwiched andfixed between the first member 1110 and the second member 1120.

Each of the first member 1110 and the second member 1120 is a basematerial for sandwiching and fixing the culture membrane 1010. Each ofthe first member 1110 and the second member 1120 is a member formed in acylindrical shape. Each of the first member 1110 and the second member1120 is formed of a resin material, rubber, or the like.

The first member 1110 includes a first cylindrical portion 1111extending in a cylindrical shape in the axial direction X, a placingportion (not shown), and a side surface opening portion 1112 penetratingthe first cylindrical portion 1111 in the radial direction. The sidesurface opening portion 1112 is provided at a predetermined portion ofthe first cylindrical portion 1111 including at least a portion closerto the placing portion side (a lower side in FIG. 16) in the axialdirection X than the culture membrane 1010 placed on the placingportion. Incidentally, the placing portion may be an axial end surfaceof the first cylindrical portion 1111 facing the axial direction X.

The second member 1120 is arranged so as to be externally inserted intoa part of the first cylindrical portion 1111 of the first member 1110.The second member 1120 includes a second cylindrical portion 1121extending in a cylindrical shape in the axial direction X, a facingportion 1122 facing the placing portion of the first member 1110 in theaxial direction X, and a side surface opening portion 1123 penetratingthe first cylindrical portion 1111 in the radial direction. The facingportion 1122 is formed in an annular shape on an inner peripheral sideof one axial end (the lower end in FIG. 16) of the second cylindricalportion 1121. The facing portion 1122 abuts against the support frame1012 of the culture membrane 1010 at an axial end surface to hold theculture membrane 1010. Incidentally, the facing portion 1122 may be anaxial end surface of the second cylindrical portion 1121 facing theaxial direction X.

The side surface opening portion 1123 is provided at a portion of thesecond cylindrical portion 1121 in the peripheral direction, and isprovided at a predetermined portion of the second cylindrical portion1121. The side surface opening portion 1123 communicates with the sidesurface opening portion 1112 of the first member 1110. The predeterminedportion at which the side surface opening portion 1123 is providedincludes at least a portion closer to the placing portion side (thelower side in FIG. 16) in the axial direction X than the culturemembrane 1010 placed on the placing portion. The side surface openingportion 1123 has, together with the side surface opening portion 1112 ofthe first member 1110, an air venting function of discharging airaccumulated on the lower surface of the culture membrane 1010 placed onthe placing portion to the outside.

The first member 1110 and the second member 1120 each have a lockingportion which restricts a relative movement between the first member1110 and the second member 1120 in the axial direction X by which thefirst member 1110 and the second member 1120 are separated from eachother in an assembled state thereof. That is, the first member 1110includes a convex portion 1113 as the locking portion, and the secondmember 1120 includes a concave portion 1124 serving as the lockingportion. The convex portion 1113 and the concave portion 1124 restrictthe relative movement between the first member 1110 and the secondmember 1120 in the axial direction X by which the first member 1110 andthe second member 1120 are separated from each other in an assembledstate of the first member 1110 to the second member 1120.

The convex portion 1113 is a protruding portion provided on an outersurface of the first cylindrical portion 1111 so as to protrude radiallyoutward. The convex portions 1113 are provided at two peripheralpositions (for example, different positions by 180°) of the first member1110, for example. The convex portion 1113 is formed to have a sizecorresponding to the concave portion 1124. Specifically, the convexportion 1113 has a width corresponding to a width of the concave portion1124.

The concave portion 1124 is a groove portion provided on an innersurface of the second cylindrical portion 1121 so as to be recessedradially outward. The concave portion 1124 is formed at one axial endportion of the second cylindrical portion 1121. The concave portions1124 are provided at two peripheral positions (for example, differentpositions by 180°) of the second member 1120, for example. The concaveportion 1124 is formed to be inclined with respect to the axialdirection X. That is, the concave portion 1124 is formed in a spiralshape so as to extend in the axial direction X while extending in theperipheral direction. The innermost portion of the concave portion 1124forms a locking wall to which the convex portion 1113 of the firstmember 1110 is locked.

The assembling of the first member 1110 and the second member 1120 isperformed such that the convex portion 1113 moves along the concaveportion 1124. The convex portion 1113 can be fitted into the innermostportion of the concave portion 1124 and locked to the locking wall. Thefirst member 1110 and the second member 1120 are assembled to each otherby fitting the convex portion 1113 into the concave portion 1124 andbeing locked to the locking wall, thereby restricting the relativemovement between the first member 1110 and the second member 1120 in theaxial direction X by which the first member 1110 and the second member1120 separated from each other.

Next, assembling of the cell culture device 1100 will be described.

The culture membrane 1010, the first member 1110, and the second member1120, which are components of the cell culture device 1100, areprepared. The cell culture device 1100 is assembled manually by a personusing a jig such as a tweezer. In an assembling procedure, first, theculture membrane 1010 is assembled to the first member 1110. The culturemembrane 1010 is gripped with a jig, the culture membrane 1010 is thenmoved in the axial direction X with respect to the first member 1110 andplaced so as to abut against the axial end surface of the placingportion of the first member 1110, thereby realizing the assembling ofthe culture membrane 1010 to the first member 1110. Incidentally, theassembling of the culture membrane 1010 to the first member 1110described above may be performed in a state in which the first member1110 is set with the axial end portion thereof on the placing portionside being positioned above.

Next, as described above, the second member 1120 is assembled to thefirst member 1110 on which the culture membrane 1010 is placed on theplacing portion. First, with a jig, the second member 1120 is externallyinserted into the first member 1110 by being moving in a directionapproaching each other in the axial direction X and is rotated in theperipheral direction, and the convex portion 1113 of the first member1110 is moved along the concave portion 1124 of the second member 1120,thereby realizing the assembling. Incidentally, the assembling of thesecond member 1120 to the first member 1110 described above may beperformed by inserting the second member 1120 from above the firstmember 0 in a state in which the first member 1110 is set with the axialend portion thereof on the placing portion side being positioned above.

When the movement is performed and the convex portion 1113 of the firstmember 1110 is fitted into the innermost portion of the concave portion1124 of the second member 1120, the relative movement of the two members1110, 1120 in the axial direction X by which the two members 1110, 1120are separated from each other is restricted, and the two members 1110,1120 are mounted to each other, so that the assembling of the twomembers 1110, 1120 is completed. When the assembling of the two members1110, 1120 is completed, the placing portion and the facing portion 1122sandwich the culture membrane 1010 (specifically, the support frame1012) therebetween, and the convex portion 1113 of the first member 1110is locked to the locking wall of the concave portion 1124 of the secondmember 1120.

According to the structure of the cell culture device 1100, after thefirst member 1110 and the second member 1120 are assembled, the relativemovement between the first member 1110 and the second member 1120 in theaxial direction X can be restricted by the convex portion 1113 and theconcave portion 1124 as the locking portions in a state in which thesupport frame 1012 of the culture membrane 1010 is sandwiched betweenthe first member 1110 and the second member 1120. Therefore,adhesiveness in the assembled state of the first member 1110 and thesecond member 1120 sandwiching the culture membrane 1010 can beimproved, and a sealing property on both sides sandwiching the culturemembrane 1010 during the cell culture can be ensured.

After the assembling of the first member 1110 and the second member 1120is completed as described above, the cell culture is performed on bothsurfaces of the culture membrane 1010 in the same manner as in the thirdembodiment. Therefore, according to the cell culture device 1100, thesame effects as those of the cell culture device 1001 of the thirdembodiment can be obtained.

In the fourth embodiment, the convex portion 1113 of the first member1110 and the concave portion 1124 of the second member 1120 eachcorrespond to the “locking portion” described in the claims.

In the fourth embodiment, the first member 1110 includes the convexportion 1113 provided on the outer surface of the first cylindricalportion 1111 so as to protrude radially outward, and the second member1120 includes the concave portion 1124 provided on the inner surface ofthe second cylindrical portion 1121 so as to be recessed radiallyoutward. However, the present invention is not limited to this, and thefirst member 1110 may have a concave portion provided on the outersurface of the first cylindrical portion 1111 so as to be recessedradially inward, and the second member 1120 may have a convex portionprovided on inner surface of the second cylindrical portion 1121 so asto protrude radially inward.

Fifth Embodiment

Similarly to the cell culture device 1001 of the third embodiment, acell culture device 1200 of a fifth embodiment is a device for culturinganchorage-dependent cells. Hereinafter, in the cell culture device 1200,the same components as those of the cell culture device 1001 are denotedby the same reference numerals, and a description thereof will beomitted or simplified.

As shown in FIGS. 17 and 18. the cell culture device 1200 includes theculture membrane 10, a first member 1210, and a second member 1220. Inthe cell culture device 1200, the culture membrane 1010 is sandwichedand fixed between the first member 1210 and the second member 1220.

Each of the first member 1210 and the second member 1220 is a basematerial for sandwiching and fixing the culture membrane 1010. Each ofthe first member 1210 and the second member 1220 is a member formed in acylindrical shape. Each of the first member 1210 and the second member1220 is formed of a resin material, rubber, or the like.

The first member 1210 includes a first cylindrical portion 1211extending in a cylindrical shape in the axial direction X and a placingportion 1212 on which the culture membrane 1010 is placed, similar tothe first cylindrical portion 1021 and the placing portion 1022 of thefirst member 1020 of the third embodiment. The second member 1220 isinserted and arranged in the first cylindrical portion 1211 of the firstmember 1210, similar to the second cylindrical portion 1031 and thefacing portion 1032 of the second member 1030 of the third embodiment.The second member 1220 includes a second cylindrical portion 1221extending in a cylindrical shape in the axial direction X, and a facingportion 1222 facing the placing portion 1212 of the first member 1210 inthe axial direction X.

The first member 1210 and the second member 1220 each have a lockingportion which restricts a relative movement in the axial direction X bywhich the first member 1210 and the second member 1220 are separatedfrom each other in an assembled state thereof. The locking portion is aso-called snap-fit type locking portion. That is, the first member 1210includes an elastic deformation portion 1213 as the locking portion, andthe second member 1220 includes a groove portion 1223 as the lockingportion. The elastic deformation portion 1213 and the groove portion1223 restrict the relative movement in the axial direction X by whichthe first member 1210 and the second member 1220 are separated from eachother in the assembled state of the first member 1210 to the secondmember 1220.

The elastic deformation portion 1213 is a portion extending in a plateshape from an axial end surface of the first cylindrical portion 1211 inthe axial direction X. The elastic deformation portion 1213 can beelastically deformed in the radial direction. The elastic deformationportion 1213 is provided at two peripheral positions (for example,different positions by 180°) of the first member 1210, for example. Aclaw portion 1214 is provided at an axial tip end portion of the elasticdeformation portion 1213. The claw portion 1214 is a hooking portionprotruding radially inward from a plate-shaped main body of the elasticdeformation portion 1213. A radially inner surface of the claw portion1214 forms an inclined surface 1215 inclined in a tapered shape. Aradial width of the claw portion 1214 is set to be the maximum at a rootside and gradually decrease toward an axial tip end.

The groove portion 1223 is provided on an outer surface of the secondcylindrical portion 1221 so as to be recessed radially inward. Thegroove portion 1223 is formed in the middle of the second cylindricalportion 1221 in the axial direction. The groove portion 1223 is aportion where the claw portion 1214 is hooked when the first member 1210and the second member 1220 approach each other in the axial direction Xto a predetermined positional relationship. The groove portions 1223 areprovided at two peripheral positions (for example, different positionsby 180°) of the second member 1220, for example. The groove portion 1223is formed to have a size corresponding to the claw portion 1214 of theelastic deformation portion 1213 of the first member 1210. The grooveportion 1223 forms a locking wall to which the claw portion 1214 of thefirst member 1210 is hooked and locked.

As shown in FIGS. 17 and 18, the assembling of the first member 1210 andthe second member 1220 is performed by relatively moving the firstmember 1210 and the second member 1220 in the axial direction X suchthat the claw portion 1214 of the elastic deformation portion 1213 ishooked by the groove portion 1223. The first member 1210 and the secondmember 1220 are assembled to each other by the claw portion 1214 beinghooked and locked to the groove portion 1223, thereby restricting therelative movement between the first member 1210 and the second member1220 in the axial direction X by which the first member 1210 and thesecond member 1220 are separated from each other.

Next, assembling of the cell culture device 1200 will be described.

The culture membrane 1010, the first member 1210, and the second member1220, which are components of the cell culture device 1200, areprepared. The cell culture device 1200 is assembled manually by a personusing a jig such as a tweezer. In an assembling procedure, first, theculture membrane 1010 is assembled to the first member 1210. The culturemembrane 1010 is gripped with a jig, the culture membrane 1010 is thenmoved in the axial direction X with respect to the first member 1210 andplaced so as to abut against the axial end surface of the placingportion 1212 of the first member 1210, thereby realizing the assemblingof the culture membrane 1010 to the first member 1210. Incidentally, theassembling of the culture membrane 1010 to the first member 1210described above may be performed in a state in which the first member1210 is set with the axial end portion thereof on the placing portionside being positioned above.

Next, as described above, the second member 1220 is assembled to thefirst member 1210 on which the culture membrane 1010 is placed on theplacing portion. The second member 1120 is inserted into the firstmember 1110 by being moved in a direction approaching each other in theaxial direction X with a jig, thereby realizing the assembling.Specifically, first, the second cylindrical portion 1221 of the secondmember 1220 abuts against the inclined surface 10215 of the claw portion1214 of the elastic deformation portion 1213 of the first member 1210,then, the axial tip end portion of the elastic deformation portion 1213is bent outward in the radial direction and deformed, thereafter, theaxial tip end portion of the elastic deformation portion 1213 isreturned radially inward when the claw portion 1214 of the elasticdeformation portion 1213 reaches the groove portion 1223 of the secondmember 1220, and the claw portion 1214 is hooked to the groove portion1223. Incidentally, the assembling of the second member 1220 to thefirst member 1210 described above may be performed by inserting thesecond member 1220 from above the first member 1210 in a state in whichthe first member 1210 is set with the axial end portion thereof on theplacing portion 1212 side positioned below.

When the movement is performed and the claw portion 1214 of the elasticdeformation portion 1213 of the first member 1210 is hooked by thegroove portion 1223 of the second member 1220, the relative movement ofthe two members 1210, 1220 in the axial direction X by which the twomembers 1210, 1220 are separated from each other is restricted, and thetwo members 1210, 1220 are mounted to each other, so that the assemblingof the two members 1210, 1220 is completed. When the assembling of thetwo members 1210, 1220 is completed, the placing portion 1212 and thefacing portion 1222 sandwich the culture membrane 1010 (specifically,the support frame 1012) therebetween, and the claw portion 1214 of thefirst member 1210 is locked to the groove portion 1223 of the secondmember 1220.

According to the structure of the cell culture device 1200, after thefirst member 1210 and the second member 1220 are assembled, the relativemovement between the first member 1210 and the second member 1220 in theaxial direction X can be restricted by the elastic deformation portion1213 and the groove portion 1223 as the locking portions in a state inwhich the support frame 1012 of the culture membrane 1010 is sandwichedbetween the first member 1210 and the second member 1220. Therefore,adhesiveness in the assembled state of the first member 1210 and thesecond member 1220 sandwiching the culture membrane 1010 can beimproved, and a sealing property on both sides sandwiching the culturemembrane 1010 during the cell culture can be ensured.

After the assembling of the first member 1210 and the second member 1220is completed as described above, the cell culture is performed on bothsurfaces of the culture membrane 1010 in the same manner as in the thirdembodiment. Therefore, according to the cell culture device 1200, thesame effects as those of the cell culture device 1001 of the thirdembodiment can be obtained. In addition, according to the cell culturedevice 1200, since the assembling of the first member 1210 and thesecond member 1220 can be realized only by moving the two members 1210,1220 in the axial direction X, assemblability can be simplified and anassembling work can be facilitated.

In order to release the assembling of the first member 1210 and thesecond member 1220, as shown in FIG. 18, the elastic deformation portion1213 of the first member 1210 may be forcibly bent outward in the radialdirection and deformed using a rod-shaped jig 1300, and in this state,the second member 1220 may be pulled out in the axial direction X fromthe first member 1210. In addition, in order to release the assemblingof the first member 1210 and the second member 1220 using the jig 1300,a part of the inclined surface 1215 needs to be positioned radiallyoutward of the outer surface of the second cylindrical portion 1221 ofthe second member 1220 so that the jig 1300 can abut against theinclined surface 1215 of the claw portion 1214, in a state in which theclaw portion 1214 of the elastic deformation portion 1213 of the firstmember 1210 is hooked by the groove portion 1223 of the second member1220. According to this configuration, the assembling of the firstmember 1210 and the second member 1220 can be released using the jig1300.

In the fifth embodiment, the elastic deformation portion 1213 of thefirst member 1210 and the groove portion 1223 of the second member 1220each correspond to the “locking portion” described in the claims.

In the fifth embodiment, the first member 1210 includes the elasticdeformation portion 1213 provided with the claw portion 1214 protrudingradially inward from the plate-shaped main body, and the second member1220 includes the groove portion 1223 provided on the outer surface ofthe second cylindrical portion 1221 so as to be recessed radiallyinward. However, the present invention is not limited to this, and thefirst member 1210 may have a groove portion provided on the innersurface of the first cylindrical portion 1211 so as to be recessedradially outward, and the second member 1220 may have an elasticdeformation portion provided with a claw portion protruding radiallyoutward from the plate-shaped main body.

In the third, fourth, and fifth embodiments described above, the culturemembrane 1010 includes the membrane body 1011 and the annular supportframe 1012 joined to the peripheral edge of the membrane body 1011.However, the present invention is not limited to this, and the supportframe 1012 may be formed in, for example, a cross shape or a net shapewhen viewed from the axial direction X as long as the support frame 1012has a frame-like structure joined to the membrane body 1011.

The present invention is not limited to the above-described embodimentsand modifications, and various changes can be made without departingfrom the spirit of the present invention.

According to an aspect of the invention, there is provided a cellculture device comprising: a holding member including a main bodycylindrical portion extending in a cylindrical shape in an axialdirection and a holding portion provided at an axially intermediateportion of the main body cylindrical portion and holding a culturemembrane; a first cylindrical member detachably mounted on one axialside with respect to the holding member, extending in a cylindricalshape in the axial direction, and protruding from one axial end of theholding member toward the one axial side when being mounted to theholding member; and a second cylindrical member detachably mounted onanother axial side with respect to the holding member, extending in acylindrical shape in the axial direction, and protruding from anotheraxial end of the holding member toward the other axial side when beingmounted to the holding member.

According to this configuration, when two types of cells are seeded onboth surfaces of the culture membrane, the cell culture device can beused in a state of removing either of the first cylindrical member andthe second cylindrical member which is positioned on a lower side of theholding member from the holding member without requiring both the firstcylindrical member and the second cylindrical member to be mounted tothe holding member and them to be used at the same time. In addition,the first cylindrical member protrudes from one axial end of the holdingmember toward the one axial side in a state of being engaged with andfixed to the holding member, and the second cylindrical member protrudesfrom the other axial end of the holding member toward the other axialside in a state of being engaged with and fixed to the holding member.Therefore, according to the cell culture device, since a dimensionaldistance from an axial lower end to the holding portion holding theculture membrane can be shortened when the cell culture is performed,cells can be cultured on the culture membrane with a small amount ofculture solution.

According to an aspect of the invention, there is also provided a cellculture device comprising: a culture membrane including a membrane bodyand a support frame joined to the membrane body; a first memberincluding a first cylindrical portion extending in a cylindrical shapein an axial direction and a placing portion which is formed with anaxial end surface facing in the axial direction, which is configured toabut against the support frame, and on which the culture membrane isplaced; and a second member including a second cylindrical portionextending in a cylindrical shape in the axial direction and a facingportion which faces the axial end surface of the placing portion in theaxial direction, the facing portion and the placing portion sandwichingthe support frame therebetween, wherein the first member and the secondmember each have a locking portion which is configured to restrict arelative movement between the first member and the second member in theaxial direction by which the first member and the second member areseparated from each other in a state in which the placing portion andthe facing portion are assembled to each other so as to sandwich thesupport frame therebetween.

According to this configuration, the locking portion can restrict arelative movement between the first member and the second member in theaxial direction by which the first member and the second member areseparated from each other in a state in which the placing portion of thefirst member and the facing portion of the second member are assembledto each other so as to sandwich the support frame of the culturemembrane therebetween. Therefore, adhesiveness in the assembled statebetween the first member and the second member sandwiching the culturemembrane can be improved, and a sealing properly between on both sidessandwiching the culture membrane during cell culture can be ensured.

What is claimed is:
 1. A cell culture device comprising: a holdingmember including a main body cylindrical portion extending in acylindrical shape in an axial direction and a holding portion providedat an axially intermediate portion of the main body cylindrical portionand holding a culture membrane; a first cylindrical member detachablymounted on one axial side with respect to the holding member, extendingin a cylindrical shape in the axial direction, and protruding from oneaxial end of the holding member toward the one axial side when beingmounted to the holding member; and a second cylindrical memberdetachably mounted on another axial side with respect to the holdingmember, extending in a cylindrical shape in the axial direction, andprotruding from another axial end of the holding member toward the otheraxial side when being mounted to the holding member.
 2. The cell culturedevice according to claim 1, wherein the holding member includes a sidesurface opening portion provided so as to penetrate, in a radialdirection, a predetermined portion of the main body cylindrical portionincluding at least a portion of the holding portion on one axial side.3. The cell culture device according to claim 2, wherein the sidesurface opening portion includes a first side surface opening portionformed at one axial side of the holding portion so as to extend in theaxial direction, and a second side surface opening portion formed atanother axial side of the holding portion so as to extend in the axialdirection.
 4. The cell culture device according to claim 1, wherein theholding member includes a first holding cylinder having a cylindricalshape in which a first abutment portion configured to abut against anaxial end surface on one axial side of the culture membrane is providedas the holding portion, and a second holding cylinder having acylindrical shape which is engageably attached to the first holdingcylinder and in which a second abutment portion configured to abutagainst an axial end surface on another axial side of the culturemembrane is provided as the holding portion.
 5. The cell culture deviceaccording to claim wherein the culture membrane includes a membrane bodyand a support frame which is joined to the membrane body and formed of amaterial harder than the membrane body.
 6. A method of using the cellculture device according to claim 1, the method comprising: mounting thefirst cylindrical member on the one axial side of the holding memberbefore cells are seeded on one axial end surface of the culturemembrane: removing the first cylindrical member from the holding memberafter the cells are seeded on the one axial end surface of the culturemembrane; and mounting the second cylindrical member on the other axialside of the holding member before cells are seeded on another axial endsurface of the culture membrane.
 7. A cell culture device comprising: aculture membrane including a membrane body and a support frame joined tothe membrane body; a first member including a first cylindrical portionextending in a cylindrical shape in an axial direction and a placingportion which is formed with an axial end surface facing in the axialdirection, which is configured to abut against the support frame, and onwhich the culture membrane is placed; and a second member including asecond cylindrical portion extending in a cylindrical shape in the axialdirection and a facing portion which faces the axial end surface of theplacing portion in the axial direction, the facing portion and theplacing portion sandwiching the support frame therebetween, wherein thefirst member and the second member each have a locking portion which isconfigured to restrict a relative movement between the first member andthe second member in the axial direction by which the first member andthe second member are separated from each other in a state in which theplacing portion and the facing portion are assembled to each other so asto sandwich the support frame therebetween.
 8. The cell culture deviceaccording to claim 7, wherein the locking portion includes a concaveportion which is provided in one of the first cylindrical portion andthe second cylindrical portion and extends in a peripheral direction,and a convex portion which is provided on the other of the firstcylindrical portion and the second cylindrical portion, and which isconfigured to relatively move in the concave portion and fit into theconcave portion by a relative rotation of the first member and thesecond member in the peripheral direction.
 9. The cell culture deviceaccording to claim 8, wherein the concave portion is provided on aperipheral wall on the one of the first cylindrical portion and thesecond cylindrical portion so as to be recessed in a radial direction,and the convex portion is provided on a peripheral wall on the other oneof the first cylindrical portion and the second cylindrical portion soas to protrude in the radial direction.
 10. The cell culture deviceaccording to claim 7, wherein the locking portion includes an elasticdeformation portion which is provided on one of the first cylindricalportion and the second cylindrical portion, which is provided with aclaw portion protruding radially at an axial tip end portion, and whichis elastically deformable in a radial direction, and a groove portionwhich is provided on the other of the first cylindrical portion and thesecond cylindrical portion, and to which the claw portion is hooked whenthe first member and the second member approach each other in the axialdirection to a predetermined positional relationship.
 11. The cellculture device according to claim 7, wherein the first member includes aside surface opening portion which is provided so as to penetrate, in aradial direction, a predetermined portion of a peripheral wall of thefirst cylindrical portion including at least a portion closer to theplacing portion in the axial direction than the culture membrane placedon the placing portion, and which opens a part of the first cylindricalportion.
 12. The cell culture device according to claim 7, wherein thefirst cylindrical portion includes a peripheral wall portion whose innerperipheral surface is formed so as to be able to guide the culturemembrane to the placing portion, and the peripheral wall portionincludes a cutout portion penetrating in a radial direction andextending in the axial direction from an axial end portion of the firstcylindrical portion toward a portion where the placing portion isprovided.
 13. The cell culture device according to claim 11, wherein thefirst cylindrical portion includes a peripheral wall portion whose innerperipheral surface is formed so as to be able to guide the culturemembrane to the placing portion, the peripheral wall portion includes acutout portion penetrating in a radial direction and extending in theaxial direction from an axial end portion of the first cylindricalportion toward a portion where the placing portion is provided, and theside surface opening portion and the cutout portion are provided at thesame position in a peripheral direction on the first member and arearranged so as to be continuous with each other in the axial direction.14. The cell culture device according to claim 11, wherein the firstcylindrical portion includes a peripheral wall portion whose innerperipheral surface is formed so as to be able to guide the culturemembrane to the placing portion, the peripheral wall portion includes acutout portion penetrating in a radial direction and extending in theaxial direction from an axial end portion of the first cylindricalportion toward a portion where the placing portion is provided, and theside surface opening portion and the cutout portion are provided atdifferent positions in a peripheral direction on the first member.